L-822492 is a highly selective and high affinity antagonist for substance P at the rat neurokinin-1 receptor (NK1R). Earlier work has shown that substance P and NK1R are involved in alcohol-related behaviors. We have previously reported that NK1R antagonism attenuates stress-induced reinstatement of alcohol seeking and suppresses escalated alcohol self-administration, but does not affect primary reinforcement or cue-induced reinstatement. We administered L-821429 or vehicle prior to footshock-induced reinstatement of alcohol seeking, and mapped the resulting neuronal activation using Fos immunohistochemistry. As expected, vehicle treated animals exposed to footshock showed induction of Fos immunoreactivity in several regions of the brain stress circuitry, including the amygdala (AMG), nucleus accumbens (NAC), dorsal raphe nucleus (DR), prefrontal cortex (PFC), and bed nucleus of the stria terminalis (BNST). NK1R antagonism selectively suppressed the stress-induced increase in Fos in the DR and NAC shell. In the DR, Fos-induction by stress largely overlapped with tryptophan hydroxylase (TrpH), indicating activation of serotonergic neurons. Of NAC shell neurons activated during stress-induced reinstatement of alcohol seeking, about 30% co-expressed dynorphin (DYN), while 70% co-expressed enkephalin (ENK). Few (<1%) activated NAC shell neurons coexpressed choline acetyltransferase (ChAT), which labels the cholinergic interneurons of this region. Infusion of the NK1R antagonist L-822429 into the NAC shell blocked stress-induced reinstatement of alcohol seeking. In contrast, L-822429 infusion into the DR had no effect, suggesting that the influence of NK1R signaling on neuronal activity in the DR is indirect. Taken together, these results outline a potential pathway through which endogenous NK1R activation mediates stress-induced alcohol seeking. We also examined the effect of L-822429, on alcohol intake and seeking behaviour in genetically selected Marchigian Sardinian alcohol preferring rats. These rats demonstrate an anxious phenotype and are highly sensitive to stress and stress-induced drinking. Systemic administration of L-822429 significantly reduced operant alcohol self-administration in Marchigian Sardinian alcohol preferring rats, but did not reduce alcohol self-administration in stock Wistar rats. NK1 receptor antagonism also attenuated yohimbine-induced reinstatement of alcohol seeking at all doses tested but had no effect on cue-induced reinstatement of alcohol seeking. cL-822429 reduced operant alcohol self-administration when injected into the lateral cerebroventricles or the medial amygdala. L-822429 injected into the medial amygdala also significantly reduced anxiety-like behavior in the elevated plus maze test. No effects on alcohol intake were observed following injection of L-822429 into the dorsal or the ventral hippocampus. These results suggest that NK1 receptor antagonists may be useful for the treatment of alcohol addiction associated with stress or comorbid anxiety disorders. The medial amygdala appears to be an important brain site of action of NK1 receptor antagonism.